METABOLISMO, MITOCONDRIA Y CÁNCER:

Master Oncología Molecular

José M. Cuezva
Departamento de Biología Molecular, Universidad Autónoma de Madrid,
Centro de Biología Molecular “Severo Ochoa”, CSIC-UAM,
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII.
Instituto de Investigación Hospital 12 de Octubre, i+12
USO EXCLUSIVO MOM
 The role of mitochondria in cell
physiology

Energy provision
Execution of cell death
Ca2+ and ROS Signaling

genetic epigenetic

Ageing

Mitochondrial
CANCER
dysfunction

Diabetes

Neurodegenerative Encephalomyopathies
diseases USO EXCLUSIVO MOM
Overview of energy metabolism:

Sánchez-Aragó et al., Antioxid. Redox Signal. 2013

USO EXCLUSIVO MOM
Metabolic reprogramming: an essential feature of cellular proliferation:

Fatty acids

Vander Heiden, M.G. Nature Reviews 10, 671-684, 2011

USO EXCLUSIVO MOM
Metabolic reprogramming in the growth phase of T lymphocytes:

Wang & Green. Nature Immunology, 13, 907-915, 2012

USO EXCLUSIVO MOM
 Metabolism of T lymphocytes:

Wang et al., Nature 261:702-705 (1976)

OXPHOS
Proliferating

Quiescent

Wang & Green. Immunological Reviews, 249, 14-26, 2012

Brand & Hermfisse, FASEB J. 11: 388-395 (1997)

USO EXCLUSIVO MOM

Metabolic reprogramming of energy provision pathways:

Vander Heiden et al., Science 324, 1029-1033, 2009

USO EXCLUSIVO MOM

Genetic alterations/mechanisms that contribute to the Warburg phenotype

Glycolytic enzymes &

glucose transporters GLYCOLYSIS

nDNA & Hypoxic

MYC AKT p53 HIF1
mtDNA microenvironment
mutations

Low OXPHOS

USO EXCLUSIVO MOM

 Glycolysis:
- cytoplasm

- 10 different enzymes:
hexokinase, phosphofructokinase,
glyceraldehyde-3-phosphate
dehydrogenase, pyruvate kinase

- 2 ATP, 2 NADH

- Metabolic intermediates

Figure 13-3 Essential Cell Biology (© Garland Science 2010) USO EXCLUSIVO MOM
 Destinos metabólicos del piruvato y del NADH:

NADH + H+ NAD+ Lactato Glucólisis anaerobia

Glucólisis

Piruvato O2

2 ATP

OH-
Lanzaderas
Piruvato NAD+
+
NAD+ Co A
CO2

Acetil-Co A + NADH
Metabolismo mitocondrial

USO EXCLUSIVO MOM

Ciclo de los ácidos tricarboxílicos:
1 Citrato sintasa
2a y 2b Aconitasa
3 Isocitrato deshidrogenasa
4 -cetoglutarato deshidrogenasa
5 Succinil-CoA sintetasa
6 Succinato deshidrogenasa
7 Fumarasa
8 Malato deshidrogenasa

Aquí se termina con

el esqueleto carbonado
de la glucosa

USO EXCLUSIVO MOM

Transporte electrónico y fosforilación oxidativa:
- Generar un gradiente de H+ (Δp) a ambos lados de la m.i.m.
- Utilizar Δp para la síntesis de ATP

NADH CI

FADH2 CII Q

CIII

Cyt c

CIV

1/2 O2 + 2H+ H2O

USO EXCLUSIVO MOM
Glycolysis and the TCA provide metabolic precursors for the biosynthesis of macromolecules.

USO EXCLUSIVO MOM

Glycolysis occurs at elevated rates
in tumor cells

USO EXCLUSIVO MOM

The switch of protein isoforms favors anabolism in proliferating cells:

Vander Heiden et al., Cold Spring Harb. Quant. Biol. 76, 325-334, 2011

USO EXCLUSIVO MOM

Allosteric regulation of PKM2 allows anabolic metabolism:

USOGuiEXCLUSIVO
et al., Science Signaling 6, MOM
1-4, 2013
Regulation of PKM2 allows anabolic metabolism:

USO EXCLUSIVO MOM

Phosphoglycerate dehydrogenase is a growth-promoting pathway activated in cancer cells:

Amplificación 16% tumores

Mullen & DeBerardinis. Trends Endocrinol. Metab. 23, 552-559, 2012
USO EXCLUSIVO MOM
Citrate is exported to the cytoplasm for the synthesis of lipids:

DeBerardinis et al., Cell Metab. 7, 11-20, 2008

USO EXCLUSIVO MOM
 ***
Glutamine anaplerosis of the TCA cycle:

***

DeBerardinis et al., Cell Metab. 7, 11-20, 2008

*** Glutamine supports pancreatic cancer growth ……….…………….Son et al., Nature 496, 4 April 2013

*** SIRT4 has a tumor-suppressive activity and regulates ……….….Jeong et al., Cancer Cell, 4 April 2013

USO EXCLUSIVO MOM

Multiple regulatory events activate the oxidative Pentose Phosphate Pathway:

Biosynthetic purposes and

redox state (GSH)

Nucleotide biosynthesis

PKM2

Metallo and Vander Heiden, Mol. Cell 49, 388-398, 2013

USO EXCLUSIVO MOM
Reductive carboxylation of glutamine feeds lipid synthesis in hypoxia:

Vander Heiden et al., Cold Spring Harb. Quant. Biol. 76, 325-334, 2011

USO EXCLUSIVO MOM

 Hypoxia inducible factor-1 (HIF-1):
mt ROS
Hypoxia
HIF-1
Succinate
HRE
HIF-1 Targeted gene

VEGF IGF-2 Glucose Metabolism

Prolyl hydroxylation

VHL Angiogenesis
Growth/survival
Ubiquitination

Transportes: GLUT1and GLUT3

Degradation by the proteasome Glycolytic enzymes: Aldolase A and C, enolase1,

hexokinase1and 3, lactate dehydrogenase A,
phosphofructokinase L, phosphoglycerate kinase 1
Aerobiosis

USO EXCLUSIVO MOM

Proliferation and differentiation of mitochondria

Proliferation Differentiation
Astrocyte

Differentiation
Adrenal gland

Heart muscle

Fetal Neonatal
Liver development
USO EXCLUSIVO MOM
Dinámica de las mitocondrias en mitosis

USO EXCLUSIVO MOM

Dinámica mitocondrial durante los primeros minutos
de la muerte de una célula

USO EXCLUSIVO MOM

 The role of metabolism in cancer biology

- …cancer cells may have an altered Before T. After T.

mitochondrial function and this alteration

would result in the elevated rates of PET imaging of bone disease
glycolysis, that is a common feature of by 18FDG-uptake before
most tumors. and after treatment
(O. Warburg, 1926)

USO EXCLUSIVO MOM

Regulation of energy metabolism: Glucose NADPH

HK
-
Nucleotides
F26P Glucose-6-P Nucleic acids

Triacylglycerols
Fructose-6-P FBP
+ PFK - +
Fructose-1,6-P
AMP

+
Phosphoenolpyruvate Amino acids
NADPH Lactate
Proteins
LDH
PK -
Pyruvate
PDH -
Acetil-CoA
Lipid synthesis

Glutamine Citrate

Isocitrate
Glutamate
IDH
-
α- ketoglutarate
Proteins
Nucleic Acids αKGDH -
Succinil-CoA ATP

Cellular ATP provision by oxidative O2

Succinate NADH
phosphorylation defines the rate of Porphyrins
SDH H2O
glucose consumption. Heme
Fumarate ADP
FH H+-ATP synthase

Malate Electron transport

and
Oxidative Phosphorylation
Oxaloacetate
USO EXCLUSIVO MOM
 The H+-ATP synthase: A central player in the provision of
metabolic energy and in the execution of cell death

Glucose
β-F1-ATPase
GAPDH
ATP
H+
H+
H+ H
+
Pyruvate e-
H+ H+
H+
R.C. Cellular
activity
ADP
Lactate e-
CO2+H2O
ATP MCF12
MCF12

20
* ANT
20

(nmol lactate/µg protein/h)

(nmol lactate/µg protein/h)

16 16

Glycolytic flux
Glycolytic flux

12 12

8 ADP 8

4 4

0 The Bioenergetic Signature: 0

-+ : DNP -+ : OLIGO
β-F1-ATPase / GAPDH ratio
USO EXCLUSIVO MOM
Colon adenocarcinomas:
1 2 3 4 5
T N T N T N T N T N

Hsp 60

-F1-ATPase

GAPDH

1.2
-F1-ATPase / Hsp 60

*
12 3
GAPDH (a.u.)
***

BEC-Index
0.8
8 2

0.4 1

**
4

0 0 0
NT NT NT
USO EXCLUSIVO MOM
 Stage II Colon Cancer Tissue Microarrays: 104 patients
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

a
2
b
c *** + 2

-F1-ATPase
d
e ++

Hsp 60
f
g ***
h 1
i
j
1
k

Normal Carcinoma 0 0
NT NT NT NT
NED PD NED PD
Hsp 60

2 2
***
*

BEC index
GAPDH
1 1 **
-F1-ATPase

**

0
NT NT 0 NT NT
NED PD NED PD

NED: No Evidence of Disease PD: Progressive Disease

USO EXCLUSIVO MOM
Stage II Colon Cancer Tissue Microarrays: IHC and WB 104 patients

DFS OS

p<< 0.05 p<< 0.05

-F1-ATPase expression: Low : High :

Hazard Ratio (95% CI) P

A low expression of -F1-ATPase C1 1
represents a higher risk for the patient: C2 3.3 (1.58-7.10) 0.002

USO EXCLUSIVO MOM

 Lung adenocarcinomas: 90 patients

4 IEF 6 8 Hsp 60: spot 338

57 -F1-ATPase: spot 424

35
GAPDH : spot 855

27
1.0 4
* , P < 0.001
0.8
3

Arbitrary units
0.6
10
2
0.4 * *
1
Histopathological classification: BA > BD, P < 0.04 0.2

Cellular differentiation: Well > Moderate / Poor, P < 0.003

0 0
N T N T
-F1/Hsp60 BEC index

USO EXCLUSIVO MOM

 Lung adenocarcinomas: 2D-gels/ MALDI/ WB 90 patients

Stage I Tumor size: T1 < 3 cm Negative lymph nodes: N0

Cummulative proportion surviving

1.0
1.0 1.0

0.8
0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

P=0.029 P=0.0044 P=0.05
0 0 0
0 20 40 60 80 100 120
0 20 40 60 80 100 120 0 20 40 60 80 100 120

Survival time (months) Survival time (months) Survival time (months)

High BEC index in red Low BEC index in black

Patients bearing lung tumors with a low BEC index had worse prognosis:

Fisher discriminant analysis

>>
K-ras ~35%; EGFR ~ 50%;
Predictor variables: β-F1 / Hsp 60 and GAPDH loss of Rb ~ 32%; p53 ~36%
Sensitivity > 97 %

USO EXCLUSIVO MOM

The bioenergetic signature of breast cancer: WB /IHC 101 patients

Normal Tumor

c-myc, HER/erbB
Fisher discriminant analysis cyclins, p53
Predictor variables: β-F1 / Hsp 60 and BEC-GAPDH
Sensitivity > 97 %
>> bcl-2
VEGF, HIF-1α, CD31
uPA/PAI-1
USO EXCLUSIVO MOM
 Response to chemotherapy:
Acute Myeloid Leukemia

% 3BrP-induced cell death

% 5-FU-induced cell death
50 SM 120
G

Cancer cells:
40 100

M 80 M
30
G 60
20 SM
40

10 20
p<0.01
0 0
0 1 2 3 0 1 2 3

β- F1/GAPDH ratio β- F1/GAPDH ratio

Shin et al., Cancer Res. 65, 3162-3170, 2005
Xiao X, et al. PLoS ONE 8(12): e83610 (2013) Sánchez-Aragó et al. J. Transl. Med. 9:19, 2011

70 P = 0.042

(%- 5 year disease-free survival)

Colon cancer patients
60
Ovarian cancer

5-FU treated patients

50

40

P=0.028 30

20

10

0
Low High
Hjerpe et al., BMC Clinical Pathology, 13:30, 2013 Tumor β- F1-ATPase
Lin et al., Int. J. Colorectal Dis. 23, 1223-1232, 2008
USO EXCLUSIVO MOM
Loss of the mitochondrial bioenergetic capacity underlies the glucose avidity of
carcinomas

ROI
p n + e+ + η
110 lung cancer patients

18FDG
Annihilation
e+ + e-
18FDG-PET

2γ

Cummulative proportion surviving

2.5
β-F1-ATPase / GAPDH ratio

1.0 β-F1 exp. >

SUV< 6.9
2.0 (n = 58) 89 (n = 44)
0.8

1.5 0.6

1.0 0.4 β-F1 exp. <

SUV > 6.9 89 (n = 62)
0.5 0.2 (n = 47)
P=0.048
0.0 p = 0.02 p = 0.06
0
4 6 8 10 0 10 20 30 40 50 60 0 10 20 30 40 50 60
Standarized Uptake OS (Months)
OS (Months)
Value (SUV)

López-Ríos et al., Cancer Res. 67:9013-9017 (2007).

USO EXCLUSIVO MOM
Association of survival and disease progression with chromosomal
instability: A genomic exploration of colorectal cancer
Sheffer et al., Proc. Natl. Acad. Sci. USA 2009 106,7131-7136

Oxidative Phosphorylation is the only pathway that had

significant association with patient’s survival
USO EXCLUSIVO MOM
The oxidation of lactate, pyruvate, alanine or fatty acids in mitochondria forces cell death:

Wenzel et al., Cell. Mol. Life Sci. 62, 3100-3105, 2005

USO EXCLUSIVO MOM

The tumor suppressor function of mitochondrial activity:

Adeno-infected HT29 tumors ApcMin/+ versus iPGC1α/ ApcMin/+

Induced carcinogenesis:
Induced carcinogenesis: azoxymethane/dextran sodium sulfate
azoxymethane/dextran sodium sulfate

D’Errico et al., PNAS 108, 6603-6608, 2011

USO EXCLUSIVO MOM
The H+-ATP synthase: a gate to cell death or cell survival

Cytoplasm

MAO

Intermembrane
p66
ROS space
H+
H+ H+
H+
e- cytC e-
TOM
CI e- Q e-
p66
e
- CIV TIM
ATP
CIII synthase ADP
O2
H2O ATP
NADH CII
H+

FADH2 Matrix

ROS

USO EXCLUSIVO MOM

 Oxidative phosphorylation and cell death are molecularly and functionally
integrated
OSCP
ADP + Pi

β
- Matsuyama et al., Mol Cell 1, 327 (1998)
α d - Dey & Moraes, J. Biol. Chem. 275, 7087 (2000)
ATP - Harris et al., Mol. Cell. Biol. 20, 3590 (2000)
- Gross et al., Mol. Cell. Biol. 20, 3125 (2000)
- Kim et al., Oncogene 21, 3139 (2002)
F6
- Shchepina et al., Oncogene 21, 8149 (2002)
H+
Matrix
γ
H+ - Park et al., J. Biol. Chem. 279, 7512 (2004)
ε
b - Shin et al., Cancer Res. 65, 3162 (2005)
δ
Membrane
- Tomiyama et al., J. Natl. Cancer Inst. 98, 1462 (2006)
a - Santamaría et al., Carcinogenesis 27, 925 (2006)
c14
- Hernlund et al., Mol Cancer Ther. 7, 1916 (2009)
- Sánchez-Aragó et al., J Transl Med. 9:19 (2011)
H+
H+
H+

16

% dead cells
12
8 *
4
0

Control

Stauro.
Stauro.+
Oligo.
Control Stauro. Oligo.+Stauro.

Oligo.
Inhibitors of the H+-ATP synthase delay the USO
execution of cell death
EXCLUSIVO MOM
The H+-ATP synthase forms part of the Permeability Transition Pore (PTP)
Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte
M, Glick GD, Petronilli V, Zoratti M, Szabó I, Lippe G, Bernardi P.

OSCP Dimers of mitochondrial ATP synthase form the permeability

ADP + Pi transition pore. Proc Natl Acad Sci U S A. 2013 Apr
9;110(15):5887-92. doi: 10.1073/pnas.1217823110.
β d
α
Bonora M, Bononi A, De Marchi E, Giorgi C, Lebiedzinska M,
ATP
Marchi S, Patergnani S, Rimessi A, Suski JM, Wojtala A,
Wieckowski MR, Kroemer G, Galluzzi L, Pinton P.
F6
H+
Role of the c subunit of the F0-ATP synthase in mitochondrial
H+
Matrix
ε γ permeability transition. Cell Cycle. 2013 Feb 15;12(4):674-83. doi:
b
δ 10.4161/cc.23599.
Membrane

c14 a Alavian KN, Beutner G, Lazrove E, Sacchetti S, Park HA,

Licznerski P, Li H, Nabili P, Hockensmith K, Graham M, Porter GA
Jr, Jonas EA.
H+
H+
H+
An uncoupling channel within the c-subunit ring of the F1FO ATP
synthase is the mitochondrial permeability transition pore.
Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):10580-5.

L. Formentini, M.P. Pereira, L. Sánchez-Cenizo, F. Santacatterina, J.J. Lucas,

C. Navarro, A. Martínez-Serrano and J.M. Cuezva.
In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes
metabolic preconditioning. EMBO Journal (2014) 33, 762-778.
USO EXCLUSIVO MOM
 The bioenergetic signature in cancer:

Response to chemotherapy:

70 P = 0.042
(%- 5 year disease-free survival)
Colon cancer patients

60
5-FU treated patients

50

40

30

20

10
Mol. Syst. Biol. (2014) 10: 744
0
Low High
Tumor β- F1-ATPase

Lin et al., Int. J. Colorectal Dis. 23, 1223-1232, 2008

The Bioenergetic Signature correlates with a functional glycolytic/OXPHOS ratio,

cancer cell migration and predicting drug response

USO EXCLUSIVO MOM

 Mechanisms that affect the H+-ATP synthase in cancer to promote
the Warburg phenotype

H+-ATP
synthase
Cancer cell

Down-regulation of content Inhibition of

activity

silencing β-
β-mRNA Up-regulation
mRNA
translation abundance of IF1

promoter
G3BP1,
hypermethylation
HuR… (ATP5B)

USO EXCLUSIVO MOM

-F1-ATPase mRNA translation and subcellular localization are stringently controlled
during development, the cell cycle and in carcinogenesis.

EM in situ hybridization

Subcellular localization and translation of -F1 mRNA requires two cis-acting elements:

 1.2 3’ UTR

 -F1 mRNA

…… and a complex set of mRNA interacting proteins

USO EXCLUSIVO MOM
 Post-transcriptional regulation of the bioenergetic signature
in prevalent human carcinomas

Breast cancer

USO EXCLUSIVO MOM

 Identification of human β-F1-ATPase mRNA BPs

SEARCH RESULTS
MALDI
Matched peptides shown in Bold Red
M N K L Y I G N L N E S V T P A D L E K V F A E H K I S Y S G Q F L V K S G Y A F V D C P D E H W A
M K A I E T F S G K V E L Q G K R L E I E H S V P K K Q R S R K I Q I R N I P P Q L R W E V L D S L
L A Q Y G T V E N C E Q V N T E S E T A V V N V T Y S N R E Q T R Q A I M K L N G H Q L E N H A L K
V S Y I P D E Q I A Q G P E N G R R G G F G S R G Q P R Q G S P V A A G A P A K Q Q Q V D I P L R L
L V P T Q Y V G A I I G K E G A T I R N I T K Q T Q S K I D V H R K E N A G A A E K A I S V H S T P
E G C S S A C K M I L E I M H K E A K D T K T A D E V P L K I L A H N N F V G R L I G K E G R N L K
K V E Q D T E T K I
Mascot Search Results
T I S S L Q D L T L Y N P E R T I T V K G A I E N C C R A E Q E I M K K V R E A

Mascot Search Results

Y E N D V A A M S L Q S H L I P G L N L A A V G L F P A S S S A V P P P P S S V T G A A P Y S S F M
Q A P E Q E M V Q V F I P A Q A V G A I I G K K G Q H I K Q L S R F A S A S I K I A P P E T P D S K

DHX9
V R M V I I T G P P E A Q F K A Q G R I Y G K L K E E N F F G P K E E V K L E T H I R V P A S A A G
R V I G K G G K T VPeptide
N E View
L Q N L T A A E V V V P R D Q T P D E N D Q V I V K I I G H F Y A S Q M A Q
R K I R D I L A Q V K Q Q H Q K G Q S N Q A Q A R R K

Peptide
MS/MS View of FYVHNDIFR
Fragmentation

NCL
Found in gi|5031703, Ras-GTPase-activating protein SH3-domain-binding protein
[Homo sapiens]
MS/MS Fragmentation of FYVHNDIFR
SFPQ Match to Query 2: 1209.594824 from(1210.602100,1+) intensity(0.0000)
Found in gi|5031703, Ras-GTPase-activating protein SH3-domain-binding
From data file DATA.TXT
protein
ILF3 [Homo sapiens]

Match to Query 2: 1209.594824 from(1210.602100,1+) intensity(0.0000)

From data file DATA.TXT
IMP1
G3BP MS/MS
NONO

Monoisotopic mass of neutral peptide Mr(calc): 1209.5930

Fixed modifications: Carbamidomethyl (C)

NPM

RL8 MS/MS Monoisotopic mass of neutral peptide Mr(calc): 1209.5930

Fixed modifications: Carbamidomethyl (C)

USO EXCLUSIVO MOM

G3BP1 hinders the initiation of β-F1 ATPase mRNA translation

USO EXCLUSIVO MOM

 Functional role of G3BP1 in breast cancer

G3BP is up-regulated in human

breast cancer.
High tumor G3BP1 expression is a
marker of worst prognosis

Cumulative proportion surviving

N=46

N=47

G3BP1
low
high p= 0.05

Disease-free survival (months)

Ortega et al., J. Cell Science 123, 2685-2696 (2010)

C.J. Barnes et al., Cancer Res. 62, 1251-1255 (2002)

USO EXCLUSIVO MOM

 Mechanisms that affect the H+-ATP synthase in cancer to promote
the Warburg phenotype

H+-ATP
synthase
Cancer cell

Down-regulation of content Inhibition of

activity

silencing β-
β-mRNA Up-regulation
mRNA
translation abundance of IF1

promoter
G3BP1,
hypermethylation
HuR… (ATP5B)

USO EXCLUSIVO MOM

 IF1, the ATPase Inhibitory Factor 1, is highly over-expressed in carcinomas:
Colon

Carcinomas
Lung
Breast

USO EXCLUSIVO MOM

Sánchez-Aragó et al., Oncogenesis 2013
IF1: is a physiological inhibitor of the H+-ATPase synthase

24 h 48 h
IF1 H49K Con. IF1 H49K Con.

IF1
H+
GAPDH H H
+ +
H H H
+

Glycolytic flux (fold induction)

2.5 + +

* *
2.0 *
ADP + Pi
1.5
IF1 ADP Pi
IF1
ATP
1.0 ATP
H+ H+
H+ H+ H+ 0.5 H
+

ATP HYDROLASE 0 ATP SYNTHASE

Control IF1 H49K - +
oligomycin

Sánchez-Cenizo et al., J. Biol. Chem. 285, 25308-25313 (2010)

USO EXCLUSIVO MOM

Silencing of IF1 in HeLa cells triggers the activation of the ATP synthase
and the reduction of aerobic glycolysis

siCRL siIF1
IF1
β-F1
Oligomycin Sensitive Respiration

Glycolytic flux (fold induction)

6 1.5
**
(pmol O2 /min/ μg protein)

╪╪
5

4 1.0

2 0.5
**
1

0 0
siCRL siIF1
siCRL siIF1
oligomycin - + - +
USO EXCLUSIVO MOM
 IF1 over-expression triggers mitochondrial ROS signaling:
Mitochondrial ROS (a.u.)

p < 0.002 p < 0.001 CRL IF1 p < 0.001

MQ MQ
p < 0.001
DNPH - + - + - + - +
p < 0.002
-97 kDa 33

p35 carbonyls
p95

(fold control)
p70 -68 kDa
p43 -43 kDa 22
p35
-29 kDa
-21 kDa
11

0
MQ -
Tubulin
MitoQ + -- +
+ - +
MitoQ - + - + CRL IF1
CRL IF1
CRL IF1

Formentini et al., Mol. Cell 45: 731-742, 2012

USO EXCLUSIVO MOM

Mitochondrial ROS activate the NF-κB pathway in colon cancer cells:
4
NF-kB promoter activity (a.u)
p < 0.03 p < 0.001 p < 0.005
p < 0.009 ACT MAP3K1 p < 0.001
1.5
p < 0.05 p < 0.01 p < 0.01 p < 0.01
p < 0.05
1.1 1.2 1.1 1.1

TICAM1
p < 0.05

NFKBIA
0.9 1.0 0.9 0.9 0.9

TLR7
HSP90 EGR1

CFB

IL8
0.7 0.5 0.7 0.6 0.7

3 0.5 0 0.4 0.3 0.5

GAPDH FOS MQ - + - + MQ - + - + MQ - + - +
MQ - + - + MQ - + - +
CRL IF1 CRL IF1 CRL IF1 CRL IF1 CRL IF1
AKT STAT1
p < 0.05 p < 0.001 p < 0.01
p < 0.04 p < 0.01 p < 0.02 p < 0.03 p < 0.04 p < 0.05
JNK BCL3

2 1.3 1.1 1.7 1.1 1.3

MAP3K1

STAT1
0.9 1.3 0.9 1.0

BCL3
EGR1
NFKBIA NFKB2 1.0

FOS
0.8 0.7 0.9 0.6 0.8
0.5 0.5 0.3 0.5
CFB IL6 0.5
MQ - + - + MQ - + - +
MQ - + - + MQ - + - + MQ - + - +
CRL IF1

1
CRL IF1 CRL IF1 CRL IF1 CRL IF1
IL8 LTA
p < 0.04 p < 0.05 p < 0.04 p < 0.05 p < 0.05 p < 0.05
p < 0.05 p < 0.05
TLR7 LPAR1 1.1 2.0 1.7 1.1 1.7

NFKB2
1.5 1.3

LPAR1
0.9 0.9 1.3
TICAM1 TLR2

TLR2
LTA
1.0

IL6
0.7 0.9 0.7 0.9
0.5 0.5 0.5 0.5 0.5
MQ - + - +
MitoQ - + - + H O
MQ - + - + MQ - + - + MQ - + - + MQ - + - +
CRL IF1 CRL IF1 CRL IF1 CRL IF1 CRL IF1
2 2
CRL IF1 ROS
p < 0.005 p < 0.005
6

CRL
5
IκBα pS36
p-IκBα / IκBα

IF1
(fold of CRL)

Ub.
4
MitoQ: - - + + - - + + p50 RelA
3
p-IκBα
2
IκBα 1

0
p50 RelA
MitoQ - + - +
CRL IF1
Formentini et al., Mol. Cell 45: 731-742, 2012
USO EXCLUSIVO MOM
 IF1 triggers aerobic glycolysis, ROS signaling and prevents cell death in lung,
breast and ovarian cancer cells

(nmol lactate / µg protein/ h)

*
6 120

STS-induced dead cells

12
*

Mitochondrial ROS
100
LUNG 10
*

Glycolytic flux

(Fold of CRL)
#

(% of CRL)
80
(HOP62, A549) 8

Sánchez-Arago et al., Oncogenesis (2013) 2, e46

3 60
6 #
CRL IF1
-IF1 4 40 *
-βF1 2 20

0 0 0
CRL IF1 IF1+MQ CRL IF1 IF1+MQ
CRL IF1 OLIGO

(nmol lactate / µg protein/ h)

* * * #
12 2 120

STS-induced dead cells

Mitochondrial ROS
10 100
BREAST *
Glycolytic flux

(Fold of CRL)

(% of CRL)
8 80
(BT549, HS578T) #
6 1 60
CRL IF1
-IF1 4 40
-βF1 2 20
0 0 0
CRL IF1 OLIGO CRL IF1 IF1+MQ CRL IF1 IF1+MQ

#
(nmol lactate / µg protein/ h)

*
14 9 140

STS-induced dead cells

12 * * 120
Mitochondrial ROS

OVARIAN
Glycolytic flux

(Fold of CRL)

10 100

(% of CRL)
6
(OVCAR8, OVCAR3) 8 80
CRL IF1
-IF1
6 # 60
4 3
-βF1 40
2
0 0
20 *
0
CRL IF1 OLIGO CRL IF1 IF1+MQ CRL IF1 IF1+MQ
USO EXCLUSIVO MOM
IF1-mediated retrograde signaling to the nucleus promotes
cellular proliferation and survival in cancer cells

GLYCOLYSIS

IF1
OXPHOS

METABOLIC H+-ATP
REPROGRAMMING
synthase ΔΨ ROS

OXPHOS

NFkB
GLYCOLYSIS

Proliferation
Cell death

Formentini et al., Mol. Cell 45: 731-742, 2012

USO EXCLUSIVO MOM

IF1 and NFκB drive hepatocellular carcinoma

Song et al., Hepatology 2014, 60: 1659-1673

USO EXCLUSIVO MOM
IF1 and NFκB drive hepatocellular carcinoma

Song et al., Hepatology 2014, 60: 1659-1673

USO EXCLUSIVO MOM
IF1 promotes proliferation and invasion in gastric cancer

Yin et al., Biomedicine and Pharmacotherapy 70, 90-96 (2015)

USO EXCLUSIVO MOM
IF1 promotes proliferation and invasion in gastric cancer

Yin et al., Biomedicine and Pharmacotherapy 70, 90-96 (2015)

USO EXCLUSIVO MOM
 High IF1 expression predicts a better prognosis in
breast and colon cancer patients:
A Breast cancer B Colon cancer
1.0 1.0

0.8 0.8
Cumulative Proportion Surviving

Cumulative Proportion Surviving

69 30

0.6 0.6

24
0.4 0.4

0.2 0.2
8
High IF1 High IF1

Low IF1 Low IF1

0 p=0.026 p=0.0001
0

0 25 50 75 100 125 0 20 40 60
DFS (months) OS (months)

Sánchez-Arago et al., Oncogenesis (2013) 2, e46

USO EXCLUSIVO MOM
Mitochondrial complex I mutations favor breast cancer progression:

Santidrian et al., J. Clin. Invest. 123, 1068-1081 (2013)

USO EXCLUSIVO MOM
Malignant growth also depends on epigenetic factors governed by the cellular
context where a cancer cell arises

Teratoma-free mouse chimera

Blood S. Muscle Intestine

Brain Kidney Thymus
Spleen Reprod. tract Lung
Teratocarcinoma Mouse Heart Liver
blastocyst
Mintz, B. and Illmensee, K. (1975) PNAS 72, 3585 and (1976) PNAS 73, 549.

Tumor
development

Medulloblastoma Cell culture

Enucleated Normal Normal

mice oocyte blastocysts E8.5 embryos

Li et al. (2003) Cancer Res. 63, 2733-2736

USO EXCLUSIVO MOM
 The Bioenergetic Signature informs of the activity of aerobic glycolysis
and of oxidative phosphorylation:

G
a M
b c
SM 1.5

Relative cristae length (a.u)

**
**
HCT116

1.0

**
0.5

0.5 μm 0
G M SM G

SM

2.5 3.0 SM 2.5

SM
β-F1-ATPase / GAPDH ratio

βF1-ATPase/ GAPDH ratio

OL-sensitive respiration
(nmol O2 /min / 106cells)
2.0 M 2.5 2.0

2.0 1.5
1.5 M M
1.5 1.0
1.0 G G G
1.0 0.5
0.5 0.5 0
P<0.001
0 0
0 3 6 9 12 0 0.5 1.0 1.5 2.0 2.5 0 2 4 6 8 10 12 14
Glycolytic flux OL-sensitive respiration Glycolytic flux
(nmol lactate / µg protein/ h) (nmol O2 /min / 106cells) (nmol lactate / µg protein/ h)
USO EXCLUSIVO MOM
Highly glycolytic cells (G-phenotype) have a very aggressive tumor
behavior when growing in vivo:

Tumor volume (cm3)

SM+M
3 ** G SM
M 20
2 (8)
T vol.~
3,000 mm3 G
1
14
0 p = 0.004 (12)
0 10 20 30 40 50
Time (days)

USO EXCLUSIVO MOM

 Pathways and processes that distinguish the M- and G-type phenotypes
( Human Genome U133 Plus 2.0 Affymetrix Platform):

Biological Process (2387 genes)

% of identified genes p-value Benjamini p-value
RNA processing 40 1,8E-32 9,3E-29
metabolic process 21 8,8E-28 1,2E-24
regulation of cell cycle 31 4,7E-18 1,7E-15
apoptosis 27 6,5 E-14 1,7E-11
cell differentiation 21 1,3E-8 1,4E-6
mitochondrion biogenesis 38 3,1E-6 2,1E-4
cell-cell adhesion 24 2,7E-3 7,2E-2
vascular development 25 2,7E-3 7,3E-2
TCA/intermediate metabolic process 48 2,8E-3 7,3E-2
glucose metabolic process 30 3,2E-3 8,1E-2
M1 M2 M3 G3 G1 G2

DNA expression / repair. Metabolic processes Angiogenesis / invasive

qPCRs
phenotype.
8
* 12 20
*
Relative expression

7
10
* 18
16
6
5 8 14
12
*
4 6
*
10
3 8

2
4
* * 6

1
*
2 4
2
*
0 0 0
DDIT3 FOS GADD45A Nt5e PHGDH UPP1 LCN2 NRP1 VEGFA
USO EXCLUSIVO MOM
 M1

M1
M1 M2 M3 G1 G2 G3

M2
M2
PCK2

M
ARG2 M3

M3
LETM2
MRPS10
HINT2

G3
G1
NAGS
ALAS1

G1
G2
G
CLIC4
ALDH2

G2
MACROD1
G3
PINK1
REEP1
GPAM
SFXN1
SLC5A3

0
0.2
0.4
0.6
0.8
1.0
1.2
MSRB3
HIBCH
PDK3 *
PPP2R1B

ATP5G1
ACADSB
TFAM
ABCB10

ATP5G3
ATPAF1
HSPA1B
* *
MTUS1
GIP3
CYB5B
CPOX
CYP24A1
* *

MOSC1 COX7b
MTERFD3
DLD
*
1.4 Oxidative phosphorylation

MTERF
ATPAF1 NDUFAF1
COX5A
SUOX
0
0.2
0.4
0.6
0.8
1.2
1.4

1.0

ACOX
AIFM1
MCCC2
MRS2L
BDH2
TUFM
IDH3A
* *

TFAM

RG9MTD1
TIMM17A
ATP5G1
*
Biogenesis

FADH1
TIMM17

NDUFAF1
EARS2
*

TUFM

MRPL23
MRPL49
for rapid colon cancer progression.

OAT
0
0.2
0.4
0.8
1.0
1.2
1.4
1.6
1.8

0.6

MCAT
SLC25A10
MRPL13
ACAA2
AIFM1

UNG
SHMT1
* *

HSPE1
BNIP3

KIAA0101
*

ATP5G3
ALAS1

IMMPL1
Metabolism

ATP5J2
SLC27A3
*
Apoptosis and

cyb5b

PPIF
OGDHL
Repression of mitochondrial biogenesis and function: a commitment

ABCB6
IDH3A
qPCRs

UCP2
* *

-0.75 0 0.75
PDHB

USO EXCLUSIVO MOM

 AT-M AT-G AT-SM

All HCT116 tumors have the mitochondrial - βF1

phenotype of G-type cells:
- Hsp60

- GAPDH
H&E 100X CEA 400X
1.2
*
1.0

β-F1/ Hsp60 ratio

0.8 n.s

0.6
0.4
0.2
0
G M

AT-SM
AT-M
AT-G
1.4 *
1.2

β-F1/ GAPDH ratio

1.0 n.s
0.8
0.6
0.4

keratin 20 400X keratin 7 400X 0.2

0
G M

AT-SM
AT-M
AT-G
USO EXCLUSIVO MOM
 OL-sensitive respiration
(nmol O2/min/106 cells)

0
0.3
0.6
0.9

0
2
4
6
8
10
12
14
Aerobic glycolysis
(nmol lactate/µg protein/h)

OL-sensitive respiration
(nmol O2/min/106 cells)
0
0.3
0.6
0.9
The microenvironment selects cancer

0
4
8

2
6
12
14

10

Aerobic glycolysis
(nmol lactate/µg protein/h)
cells with a diminished bioenergetic function of mitochondria

USO EXCLUSIVO MOM

 “The Hallmarks of Cancer”

Energy Metabolism Ж Avoiding Immune Destruction

╩

D. Hanahan and R.A. Weinberg. Cell 100, 57-70 (2000) Cell 144, 646-674 (2011)

USO EXCLUSIVO MOM

Metabolic targets for cancer therapy:

Preclinical evidence

Prospective
clinical studies

Clinical experience

Targeting metabolism to halt anabolism or induce energy stress in cancer cells:

Galluzzi et al., Nature Reviews Grug Discovery, 12, 829-846 (2013)
USO EXCLUSIVO MOM